The present invention relates to recombinant DNA which encodes the BsmBI restriction endonuclease (endonuclease) as well as the BsmBI methyltransferase (methylase), expression of BsmBI endonuclease and methylase in E. coli cells containing the recombinant DNA.
BsmBI endonuclease is found in the strain of Bacillus stearothermophilus B61 (New England Biolabs"" strain collection #857). It recognizes the double-stranded DNA sequence 5xe2x80x2CGTCTC3xe2x80x2N1/N5 (SEQ ID NO:1) and cleaves at N1 (top strand) and N5 (bottom strand) downstream of the recognition sequence to generate a 4-base 5xe2x80x2 overhang (N=A, T, C, or G;/indicates the cleavage of phosphodiester bond). BsmBI methylase (M.BsmBI) is also found in the strain of Bacillus stearothermophilus B61. It recognizes the double-stranded DNA sequences 5xe2x80x2CGTCTC3xe2x80x2 (SEQ ID NO:2) (top strand) and 5xe2x80x2GAGACG3xe2x80x2 (SEQ ID NO:3) (bottom strand) and probably modifies a cytosine (5 mC) on the top strand and an adenosine (N6mA) on the bottom strand within the recognition sequences.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2 (SEQ ID NO:4), 5xe2x80x2PuG/GNCCPy3xe2x80x2 (SEQ ID NO:5) and 5xe2x80x2CACNNN/GTG3xe2x80x2 (SEQ ID NO:6) respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2 (SEQ ID NO:7).
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacz fusion (U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535, 1996). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (SEQ ID NO:8) (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2 (SEQ ID NO:9)) refractory to NotI digestion (New England Biolabs"" Catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the BsmBI restriction endonuclease from Bacillus stearothermophilus B61 into E. coli by methylase selection and inverse PCR. A methylase gene with high homology to C5 methylase was found in a DNA library after methylase selection. A second methylase gene with high homology to amino-methylases (N6-adenosine methylases) was found after sequencing the genomic DNA surrounding the first methylase gene. It was later determined that these two genes are fused together and code for a single fusion protein. This fusion gene was named BsmBI methylase gene (bsmBIM). The bsmBIM gene was amplified by PCR and cloned into pACYC184. The premodified host ER2744 [pACYC-BsmBIM] was used for BsmBI endonuclease expression.
It proved to be difficult to express BsmBI endonuclease in medium-copy-number expression vectors. When PCR DNA containing bsmBIR gene was ligated to pAII17 or pET21at, no active clones with correct insert were found, which may be due to under-methylation of BsmBI sites in E. coli genome or the vectors.
In order to construct a stable expression clone, the bsmBIM gene was amplified by PCR and cloned into pBR322, giving rise to pre-modified host ER2744 [pBR322-BsmBIM]. The bsmBIR gene was amplified by PCR and inserted into pACYC-T7ter with compatible ends. High BsmBI endonuclease activity was detected. However, the expression clone ER2744 [pBR-BsmBIM, pACYC-T7ter-BsmBIR] was not very stable because lower BsmBI activity was detected in larger amplified cultures. To further stabilize the expression clone, two strategies were used:
a. Introducing another plasmid carrying the T7 lysS gene coding for T7 lysozyme which inhibits T7 RNA polymerase and thus reduces the constitutive expression from the T7 promoter.
b. Using a non-cognate methylase BsmAI methylase to premodify the expression host (BsmAI methylase recognition sequence 5xe2x80x2GTCTC3xe2x80x2 N1/N5 (SEQ ID NO:27).
Two production strains were constructed. The first one was ER2566 [pBR322-BsmBIM, pCEF8, pACYC-T7ter-BsmBIR]. The second strain was a pre-modified strain by a non-cognate methylase, BsmAI methylase, ER2566 [pBR322-BsmAIM, pACYC-T7ter-BsmBIR]. This second strain generated the highest BsmBI production yield in amplified large cultures.